Because of the insufficient supply of human organs, various xenogeneic sources have been studied for transplantation (Cooper D K. Transplant Proc. 1992; 24: 2393-2396). In this regard, a pig model for transplantation has been developed. However, the use of pigs as an organ source is limited because transplantation of pigs' organs to human bodies results in various immunological rejections to the grafted organs in the human bodies, including hyperacute rejection, delay xenograft rejection (DXR) including acute vascular rejection (also called delay vascular rejection) and acute cellular rejection, and chronic rejection (Nowak R., Science 1994; 266: 1148-1151). It was found that human decay accelerating factor (hDAF, also named CD55; Cozzi et al., Transplant Proc. 1994; 26: 1402-1403 and McCurry et. al., Nat. Med. 1995; 1: 423-427), membrane cofactor protein (MCP; McCurry et. al. supra), and CD59 (McCurry et. al. supra; and Fodor et al., Proc. Natl. Acad. Sci. USA 1994; 91: 11153-11157) can down-regulate the pathway of complement activation, and thus conquers hyperacute rejection to the organs from transgenic pigs. In ex-vivo perfusion studies, hyperacute rejection was prevented in the heart (Schmoeckel et al. Transplantation 1996; 62: 729-734; and Byrne et al., Transplantation 1997; 63: 149-155), kidney (Byrne et al. Supra, Kroshus et al., Transplantation 1996; 61: 1513-1521, Storck et al., Transplantation 1997; 63: 304-310) and lung (Kroshus et al. Supra, Storck et al. Supra, and Pierson R et al., J. Heart Lung Transplant 1997; 16: 231-239) of the transgenic pig introduced with a gene encoding hDAF or CD59. It was also demonstrated that expression of a functional human CD59 on cardiac endothelium of a transgenic pig inhibits the assembling of membrane attack complex, and reduces damages on the heart tissue of a baboon, to which the heart of the transgenic pig was transplanted (Diamond et al., Transplantation 1996; 61: 1241-1249). In addition, the Imutran Limited has provided data showing that transplantation of the kidney and heart from a hDAF transgenic pig to a non-human primate recipient allows the recipient to maintain a life period of over 78 days and one month, respectively (Cozzi et al., Transplantation 2000; 70: 15-21, and Vial et al., J. Heart Lung Transplant. 2000; 19: 224-229.)
In addition, problems regarding natural antibodies generated in human recipients against the epitope of gal-alpha1, 3-gal (i.e., alpha-Gal) carried by endothelial cells of donors' organs remain to be solved. Alpha-Gal is expressed in various tissues, especially the endothelial cells of capillaries, arterioles and arteries, and also in the parencyma of liver, proximal convoluted tubules, glomerulii and collecting ducts of kidney, alveoli of lung, and pancreas ducts (Sandrin M. S. and I. F. C. McKenzie. 1999. Modulation of α-Gal epitope expression on porcine cells. In: α-Gal and anti-Gal: α-1,3-Galactosyltransferase, α-Gal epitope, and the nature anti-Gal antibody. pp. 311-337). The synthesis of the epitope of alpha-Gal depends on the activity of the enzyme, alpha1, 3-galactosyltransferase (i.e., al, 3-GT) (Galili, U. Immun. Series, 1991; 55:355-337). This enzyme is active in New World monkeys (Galili, U. et al., J. Biol. Chem. 1988; 263:17755-17762), in most species including microbias (Larsen, R. D. et al., Proc. Natl. Acad. Sci. USA. 1989; 86:8227-8231), but not in ape, Old World monkeys and human (Galili, U. supra), wherein in human the α1, 3-GT gene is present as a processed pseudogene, which is non-functional due to frame shift mutations (Larsen, R. D. et al., J. Biol. Chem. 1990; 265:7055-7061). The gene encoding α1, 3-GT has been analyzed in mice (Sandrin, M. S. et al., Xenotransplantation 1994; 1:81-88.) and pigs (Joziasse, D. H. et al., J. Biol. Chem. 266: 6991-6998). It has been suggested that organs of genetically modified pigs in which the α1, 3-GT gene is knocked out might be suitable for xenograft. Recently, α1, 3-GT genetic knockout heterozygote (Dai Y et al., Nature Biotech. 2002; 20: 251-255, and Lai L et al., Science 2002; 295: 1089-1092) and homozygote (Phelps C J et al., Science 2002; 299:411-414) pigs have been cloned. However, the applicability of the α1, 3-GT genetic knockout pigs in xenograft remains to be elucidated. Alternatively, a strategy for competitively inhibiting the alpha-Gal expression in pigs' organs by transgenesis of alpha 1, 2-fucosyltransferase was reported but failed since the alpha-Gal was still expressed with a high density in the pigs' tissues (Sharma A et al., Proc. Natl. Acad. Sci. USA 1996; 93: 7190-7195).
Therefore, there is a need to develop a transgenic animal for providing a graft to be transplanted to a human subject in need thereof, wherein one or more immunological rejections to the graft in the human subject are reduced.
Heme oxygenases (HOs), rate-limiting enzymes in heme catabolism, also named HSP32, belong to members of heat shock proteins, wherein the heme ring is cleaved into ferrous iron, carbon monoxide (CO) and biliverdin that is then converted to bilirubin by biliverdin reductase. Three isoforms of HOs, including HO-1, HO-2 and HO-3, have been cloned. The expression of HO-1 is highly inducible, whereas HO-2 and HO-3 are constitutively expressed (Maines M D et al., Annual Review of Pharmacology & Toxicology 1997; 37:517-554, and Choi A M et al., American Journal of Respiratory Cell & Molecular Biology 1996; 15:9-19). An analysis of HO-1−/− mice suggests that the gene encoding HO-1 regulates iron homeostasis and acts as a cytoprotective gene having potent antioxidant, anti-inflammatory and anti-apoptotic effects (Poss K D et al., Proceedings of the National Academy of Sciences of the United States of America 1997; 94:10925-10930, Poss K D et al., Proceedings of the National Academy of Sciences of the United States of America 1997; 94:10919-10924, and Soares M P et al., Nature Medicine 1998; 4:1073-1077). Similar findings were recently described in a case report of HO-1 deficiency in humans (Yachie A et al., Journal of Clinical Investigation 1999; 103:129-135). The molecular mechanisms responsible for the cytoprotective effects of HO-1, including anti-inflammation, anti-oxidation and anti-apoptosis, are mediated by its' reaction products.
HO-1 expression can be modulated in vitro and in vivo by protoporphyrins with different metals. Cobalt protoporphyrins (CoPP) and iron protoporphyrins (FePP) can up-regulate the expression of HO-1. In contrast, tin protoporphyrins (SnPP) and zinc protoporphyrins (ZnPP) inhibit the activity of HO-1 at the protein level. Recently, it has been proved that the expression of HO-1 suppresses the rejection of mouse-to-rat cardiac transplants (Sato K et al., J. Immunol. 2001; 166:4185-4194), protects islet cells from apoptosis, and improves the in vivo function of islet cells after transplantation (Pileggi A et al., Diabetes 2001; 50: 1983-1991). It has also been proved that administration of HO-1 by gene transfer provides protection against hyperoxia-induced lung injury (Otterbein L E et al., J Clin Invest 1999; 103: 1047-1054), upregulation of HO-1 protects genetically fat Zucker rat livers from ischemia/reperfusion injury (Amersi F et al., J Clin Invest 1999; 104: 1631-1639), and ablation or expression of HO-1 gene modulates cisplatin-induced renal tubular apoptosis (Shiraishi F et al., Am J Physiol Renal Physiol 2000; 278:F726-F736). In transgenic animal models, it was shown that over-expression of HO-1 prevents the pulmonary inflammatory and vascular responses to hypoxia (Minamino T et al., Proc. Natl. Acad. Sci. USA 2001; 98:8798-8803) and protects heart against ischemia and reperfusion injury (Yet S F, et al., Cir Res 2001; 89:168-173).
So far, no transgenic pigs carrying both hDAF and hHO-1 transgenes have been developed for xenotransplantation, and no related teachings and motivation have been provided.